Actin Tail Formation Research Articles

Mycobacterial PE/PPE proteins function as “personal protective equipment” against host defenses

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial infection worldwide, but many molecular details of how it interacts with the innate immune system remain obscure. In particular, although Mtb secretes a large number of putative effector proteins, a relatively small number have assigned functions in facilitating host-pathogen interactions. One particularly large family of secreted mycobacterial proteins that remains poorly understood is the PE/PPE proteins. Despite numerous lines of evidence for potential roles in virulence and in mediating host-pathogen interactions, only a small fraction of these 170+ proteins have been well characterized. However, this large family of proteins is likely key for understanding how Mtb subverts immune responses, manipulates host cell biology, and establishes a successful infection. Here, we highlight examples of PE/PPEs that have well-defined effects on cell intrinsic pathways in macrophages during mycobacterial infection. Examples include PPE2, which blunts production of reactive oxygen species and nitric oxide; PE_PGRS33, which facilitates bacterial uptake; PE_PGRS29, which directly binds ubiquitin to promote host autophagy and limit pathologic inflammation; MirA, which facilitates actin tail formation to promote cell-to-cell spread; and others. Understanding the full spectrum of PE/PPE functions is critical for understanding Mtb pathogenesis and for developing new strategies to combat the worldwide TB pandemic. Advancing the lagging research efforts characterizing this mysterious family of effector proteins is critical for the TB field.

Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B.thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B.thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

RACK1 promotes Shigella flexneri actin-mediated invasion, motility, and cell-to-cell spreading

Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.

Cycle-Inhibiting Factor Is Associated with Burkholderia pseudomallei Invasion in Human Neuronal Cells.

Simple SummaryNeurological melioidosis, caused by Burkholderia pseudomallei, can lead to the development of severe symptoms associated with the nervous system. However, the pathogenic mechanism through which this bacterium infects neuronal cells has not been studied. This study showed that B. pseudomallei can infect human neuronal SH-SY5Y cells in vitro. Cycle-inhibiting factor (Cif), a type III secreted effector, is one of the virulence factors produced by B. pseudomallei upon infection. The B. pseudomallei cif-deleted mutant reduced the ability to invade neuronal cells compared with the parental strain. Our finding indicates that Cif is associated with B. pseudomallei invasion of human neuronal cells.Burkholderia pseudomallei is a pathogenic bacterium that causes human melioidosis, which is associated with a high mortality rate. However, the underlying mechanisms of B. pseudomallei pathogenesis are largely unknown. In this study, we examined the infection of human neuronal SH-Sy5y cells by several clinically relevant B. pseudomallei strains. We found that all tested B. pseudomallei strains can invade SH-Sy5y cells, undergo intracellular replication, cause actin-tail formation, and form multinucleated giant cells. Additionally, a deletion mutant of B. pseudomallei cycle-inhibiting factor (cif) was constructed that exhibited reduced invasion in SH-Sy5y cells. Complementation of cif restored invasion of the B. pseudomallei cif-deleted mutant. Our findings enhance understanding of B. pseudomallei pathogenicity in terms of the virulence factor Cif and demonstrate the function of Cif in neurological melioidosis. This may eventually lead to the discovery of novel targets for treatment and a strategy to control the disease.

Regulator of Actin-Based Motility (RoaM) Downregulates Actin Tail Formation by Rickettsia rickettsii and Is Negatively Selected in Mammalian Cell Culture.

ABSTRACTThe etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is an obligately intracellular pathogen that induces the polymerization of actin filaments to propel the bacterium through the cytoplasm and spread to new host cells. Cell-to-cell spread via actin-based motility is considered a key virulence determinant for spotted fever group rickettsiae, as interruption of sca2, the gene directly responsible for actin polymerization, has been shown to reduce fever in guinea pigs. However, little is known about how, or if, motility is regulated by the bacterium itself. We isolated a hyperspreading variant of R. rickettsii Sheila Smith that produces actin tails at an increased rate. A1G_06520 (roaM [regulator of actin-based motility]) was identified as a negative regulator of actin tail formation. Disruption of RoaM significantly increased the number of actin tails compared to the wild-type strain but did not increase virulence in guinea pigs; however, overexpression of RoaM dramatically decreased the presence of actin tails and moderated fever response. Localization experiments suggest that RoaM is not secreted, while reverse transcription-quantitative PCR (RT-qPCR) data show that various levels of RoaM do not significantly affect the expression of the known rickettsial actin-regulating proteins sca2, sca4, and rickA. Taken together, the data suggest a previously unrecognized level of regulation of actin-based motility in spotted fever group rickettsiae. Although this gene is intact in many isolates of spotted fever, transitional, and ancestral group Rickettsia spp., it is often ablated in highly passaged laboratory strains. Serial passage experiments revealed strong negative selection of roaM in Vero 76 cells.

Mechanistic insight into bacterial entrapment by septin cage reconstitution

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.

Absence of BapA type III effector protein affects Burkholderia pseudomallei intracellular lifecycle in human host cells

The etiological agent of melioidosis, Burkholderia pseudomallei, utilises a type III secretion system cluster 3 (T3SS3) to deliver proteins termed type III effectors (T3SEs) into the host cytoplasm in order to establish an intracellular lifecycle in phagocytic and non-phagocytic cells, thus playing an important role in pathogenesis. To gain insight into possible functional roles for BapA, a putative T3SE with unknown function, in the intracellular lifecycle of B.pseudomallei, bapA gene knockout mutant was constructed. The effect of the knockout on virulence to the otherwise isogenic parental strain, K96243, was studied by cellular infection assays and Caenorhabditis elegans killing assay. The attachment and subsequent entry into A549 cells was significantly (P < 0.05) attenuated in the ΔbapA compared to K96243. However, intracellular replication was not affected. Furthermore, the cell-to-cell spreading capacity of ΔbapA was impaired although the mutant exhibited no evident defect in its actin tail formation. Additionally, phagocytosis and intracellular replication rates of ΔbapA in U937 macrophage cells were significantly reduced relative to K96243 without phagosomal escape being affected. Based on these observations, we conclude that the BapA T3SE could play an important role in B.pseudomallei intracellular lifecycle, especially, in the early stages of attachment and entry into the host cell.

The GBP1 microcapsule interferes with IcsA-dependent septin cage assembly around Shigella flexneri.

Many cytosolic bacterial pathogens hijack the host actin polymerization machinery to form actin tails that promote direct cell-to-cell spread, enabling these pathogens to avoid extracellular immune defenses. However, these pathogens are still susceptible to intracellular cell-autonomous immune responses that restrict bacterial actin-based motility. Two classes of cytosolic antimotility factors, septins and guanylate-binding proteins (GBPs), have recently been established to block actin tail formation by the human-adapted bacterial pathogen Shigella flexneri. Both septin cages and GBP1 microcapsules restrict S. flexneri cell-to-cell spread by blocking S. flexneri actin-based motility. While septins assemble into cage-like structures around immobile S. flexneri, GBP1 forms microcapsules around both motile and immobile bacteria. The interplay between these two defense programs remains elusive. Here, we demonstrate that GBP1 microcapsules block septin cage assembly, likely by interfering with the function of S. flexneri IcsA, the outer membrane protein that promotes actin-based motility, as this protein is required for septin cage formation. However, S. flexneri that escape from GBP1 microcapsules via the activity of IpaH9.8, a type III secreted effector that promotes the degradation of GBPs, are often captured within septin cages. Thus, our studies reveal how septin cages and GBP1 microcapsules represent complementary host cell antimotility strategies.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Shigella MreB promotes polar IcsA positioning for actin tail formation.

ABSTRACTPathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host–pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper.

First person – Sina Krokowski

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Sina Krokowski is first author on ‘Shigella MreB promotes polar IcsA positioning for actin tail formation’, published in JCS. Sina conducted the research described in this article while a PhD student in Serge Mostowy's lab at Imperial College London, UK. She is now a postdoc in the lab of Andres Floto on the Cambridge Biomedical Campus, Cambridge, UK investigating the cell biology underlying host–pathogen interactions, and specifically cell autonomous immunity towards bacterial pathogens.

Septins suppress the release of vaccinia virus from infected cells.

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility.

ABSTRACTDynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.

Vaccinia virus egress mediated by virus protein A36 is reliant on the F12 protein.

Egress of vaccinia virus from its host cell is mediated by the microtubule-associated motor kinesin-1, and three viral proteins, A36 and the F12/E2 complex, have been implicated in this process. Deletion of F12 expression causes a more severe reduction in egress than deletion of A36 but whether these proteins are involved in the same or different mechanisms of kinesin-1 recruitment is unknown. Here it is shown that a virus lacking both proteins forms a smaller plaque than mutants lacking either gene alone, indicating non-redundant functions. A36 not only links virions directly to kinesin-1 but also nucleates actin polymerization to propel surface virions away from the host cell. To address the relative importance of these functions for virus spread, a panel of recombinant viruses was constructed in which the ability of A36 to bind kinesin-1 or to nucleate actin polymerization was abrogated individually or together, in the presence or absence of F12 expression. Analysis of these viruses revealed that in the presence of the F12 protein, loss of kinesin-1 interaction made a greater contribution to plaque size than did the formation of actin tails. However in the absence of F12, the ability of A36 to promote egress was abrogated. Therefore, the ability of A36 to promote egress by kinesin-1 is reliant on the F12 protein.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.

The Shigella Virulence Factor IcsA Relieves N-WASP Autoinhibition by Displacing the Verprolin Homology/Cofilin/Acidic (VCA) Domain

Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53–758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation.

Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice.

Listeria monocytogenes is an intracellular pathogen that causes listeriosis. Due to its intracellular niche, L. monocytogenes has evolved to limit immune recognition and response to infection. Antibodies that are slightly induced by listerial infection are completely unable to protect re-infection of L. monocytogenes. Thus, a role of antibody on the protective effect against L. monocytogenes infection has been neglected for a long time. In the present study, we reported that passive immunization with an excessive amount of antibodies against ActA and listeriolysin O (LLO) attenuates severity of L. monocytogenes infection. Combination of these antibodies improved survival of L. monocytogenes infected mice. Bacterial load in spleen and liver of listerial infected mice and infected RAW264.7 cells were significantly reduced by administration of anti-ActA and anti-LLO antibodies. In addition, anti-LLO antibody neutralized LLO activity and inhibited the bacterial escape from the lysosomal compartments. Moreover, anti-ActA antibody neutralized ActA activity and suppressed actin tail formation and cell-to-cell spread. Thus, our studies reveal that passive immunization with the excessive amount of anti-ActA and -LLO antibodies has potential to provide the protective effect against listerial infection.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation.

ABSTRACTListeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function.

The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway.

ABSTRACTLegionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses.